Bovine rotavirus fusion protein and calf diarrhea polyvalent vaccine

A bovine rotavirus and fusion protein technology, which is applied in the field of bovine rotavirus fusion protein and calf diarrhea multiple vaccines, to achieve good immunogenicity, expand the scope of disease prevention and control, and save labor

Active Publication Date: 2020-08-11
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no commercially available vaccines for the prevention and control of calf diarrhea caused by multiple pathogens

Method used

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  • Bovine rotavirus fusion protein and calf diarrhea polyvalent vaccine
  • Bovine rotavirus fusion protein and calf diarrhea polyvalent vaccine
  • Bovine rotavirus fusion protein and calf diarrhea polyvalent vaccine

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preparation example Construction

[0064] In some optional embodiments, taking yeast as the host cell as an example, the preparation method includes the steps of gene preparation, recombinant vector construction, cell transfection, high-efficiency expression strain screening, and recombinant protein acquisition, specifically including: The obtained vector is linearized and electrotransformed into yeast competent cells, the fusion protein is expressed, and the fusion protein is purified to obtain the fusion protein. Then, an appropriate buffer is selected to assemble the purified fusion protein for virus-like particles, and the assembly effect is identified by electron microscope. The vector preferably uses the pPIC3.5K vector. After the linearized vector is electrotransformed into yeast competent cells, it is preferably directly coated on a high antibiotic concentration plate, and high selection pressure is used to select high expression monoclonal strains. The electrotransformation preferably uses multiple elec...

Embodiment 1

[0070] Express the fusion protein through the Pichia pastoris expression system:

[0071] By means of structural biology, the structure of the bovine rotavirus VP6 protein was analyzed, and the epitopes of bovine coronavirus and pathogenic E. coli were chimerized into the bovine rotavirus VP6 protein backbone, and the sequence was named VP6-S -K88 / K99, the specific amino acid sequence is shown in SEQ ID NO. 8;

[0072] The fusion protein VP6-S-K88 / K99 was codon optimized for Pichia pastoris expression system, and the nucleotide sequence is shown in SEQ ID NO.9.

[0073] And by means of genetic engineering, the gene encoding the VP6-S-K88 / K99 fusion protein was synthesized, and the target gene and pPIC3.5k plasmid were digested with Bam HI and AgeI respectively (restriction system: 1μg plasmid, 1μL Bam HI, 1μL Age I, 5μL CutSmart, ddH 2 O fill up to 50μL; incubate at 37°C for 1h), recover the band of interest through a 1% agarose gel (Axyprep DNA Gel Recovery Kit AP-GX-250), measure ...

Embodiment 2

[0079] To express multi-layer chimeric VLP particles through the Pichia pastoris expression system:

[0080] By means of genetic engineering, the gene encoding VP2-N93 protein was synthesized, and the amino acid sequence is shown in SEQ ID NO.10. The target gene and pPICZ A vector were digested with Bst BI and Age I respectively (restriction system: 1μg plasmid, 1μL BstBI, 1μL Age I, 5μL CutSmart, ddH 2 O fill up to 50μL; incubate at 37°C for 1h), recover the target bands by 1% agarose gel (Axyprep DNA Gel Recovery Kit AP-GX-250), and measure the concentrations respectively, and then use T4 DNA Ligase (NEB , M0202) Connect the vector to the gene, mix the ligation system with E.Coli DH5α competent cells, incubate on ice for 30 minutes, then place at 42°C for 90 seconds, then immediately place on ice for 2 minutes, and apply directly to 0.1mg / mL ampicillin Plate, culture at 37°C for 16h, pick a single clone into 2mL LB medium and culture for 16h, extract the plasmid by Axgen kit (A...

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Abstract

The invention provides a bovine rotavirus fusion protein and a calf diarrhea polyvalent vaccine, and relates to the technical field of molecular biology. The bovine rotavirus fusion protein contains aVP6 fragment, and the VP6 fragment contains an amino acid sequence as shown in SEQ ID NO.4, wherein at least one loop region in the following (a)-(c) is replaced with an epitope derived from bovine coronavirus and / or an epitope derived from escherichia coli: (a) 168th-177th amino acid residues with an amino acid sequence as shown in SEQ ID NO.1; (b) 194th-205th amino acid residues with an amino acid sequence as shown in SEQ ID NO.2; and (c) 296th-316th amino acid residues with an amino acid sequence as shown in SEQ ID NO. 3. The bovine rotavirus fusion protein contains a plurality of epitopes, and a host can generate a plurality of antibodies after the host is immunized.

Description

Technical field [0001] The invention relates to the technical field of molecular biology, in particular to a multi-linked vaccine of bovine rotavirus fusion protein and calf diarrhea. Background technique [0002] Calf diarrhea is one of the most common clinical diseases in cattle farms. It is common in calves born 7-15 days old. It has a high morbidity and mortality rate. It not only causes significant economic losses to the cattle farm, but also affects the calves. Growth and development and lactation performance in adulthood. Among pathogenic factors, bovine rotavirus (BRV), bovine coronavirus and toxin-producing E. coli are the three main pathogens causing calf diarrhea. Among them, the incidence of calf diarrhea caused by BRV is the highest, accounting for approximately 46% of calf diarrhea cases. [0003] After bovine rotavirus infection, it is clinically characterized by atrophy, anorexia, vomiting, diarrhea and dehydration, and weight loss. Bovine coronavirus is one of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/295A61K39/15A61K39/215A61K39/108A61P31/14A61P31/04A61P1/12
CPCC07K14/005C07K14/245A61K39/12A61K39/0258A61P31/14A61P31/04A61P1/12C12N2720/12322C12N2720/12323C12N2720/12352C12N2720/12334C12N2770/20022C12N2770/20034C12N2770/20023C12N2770/20052C07K2319/40A61K2039/5258A61K2039/552A61K2039/70A61K2039/55566C07K2319/00C12R2001/19Y02A50/30A61K39/15C12N7/00
Inventor 贺笋张国庆闫鹏先席娜郭苗苗肖升东聂祥祥孔楚心李延涛潘毅平
Owner 天康制药股份有限公司
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